Method for adding an apparent non-signal line to a lateral flow assay

ABSTRACT

A test device and method for determining the presence or absence of an analyte in a fluid sample, the test device including a support bearing a mark thereon, and a matrix defining an axial flow path. In operation, an observation area in the test device becomes transparent, thereby allowing the user to view a mark that is present on a support that is disposed beneath the observation area. Typically, the mark on the underlying support is configured as a minus (−) sign. In the absence of analyte in the sample, the test device presents a negative result as a minus (−) signal. In the presence of analyte in the sample, however, the mark operates in concert with a perpendicular test line on the observation area to present a positive result as a plus (+) signal that is visible to the user.

CROSS-REFERENCES TO RELATED APPLICATIONS

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BACKGROUND OF THE INVENTION

The present invention relates generally to devices and methods fordetection of analytes in test samples. More specifically, the presentinvention provides solid phase test strip devices and methods thatcombine an internal indicator on the test strip with an external marklocated on a support.

Various analytical procedures and devices are commonly employed indetection assays to determine the presence and/or amount of substance ofinterest or clinical significance which may be present in biological ornon-biological fluids. Such substances are generally termed “analytes”and can include antibodies, antigens, drugs, or hormones.

The present invention includes lateral flow chromatography assayformats. Generally, these assays have an extended base layer on which adifferentiation can be made between a sample application region and anevaluation region. In typical use, the sample is applied to the sampleapplication region, flows along a liquid transport path which runsparallel to the base layer, and then flows into the evaluation region. Acapture reagent is present in the evaluation region, and the capturedanalyte can be detected by a variety of protocols to detect visiblemoieties associated with the captured analyte. For example, the assaymay produce a visual signal, such as color change, fluorescence,luminescence, and the like, when indicating the presence or absence ofan analyte in a biological sample.

Optimally, such test devices will provide a characteristic signal whenthe analyte is present in a sample, and a different signal when theanalyte is absent from a sample. Most typically, the test device willdisplay a “plus” (+) signal in the presence of analyte, and a “minus”(−) signal in the absence of analyte. The plus/minus test result formathas enjoyed enthusiastic customer response and wide commercial success.

Test devices of this kind are well known in the art, and play animportant role in areas such as clinical chemistry. They are used byskilled clinicians and lay person alike. Thus, there is a strong impetusto provide devices that are simple and reliable. Desirably, the assaysare single-step devices wherein the user need only apply the sampleprior to viewing the result. Single-step devices obviate the necessityof performing complicated and time consuming processing steps that mayintroduce errors in the end result.

Examples of such assays include pregnancy tests, ovulation tests,various urine, saliva, spinal, and blood tests, as well as otherclinical or diagnostic assays.

Test devices typically use materials that specifically bind with ananalyte of interest. A homologous pair of specific binding pair members(“sbp members”), usually an immunological pair comprising a ligand and areceptor (antiligand), is involved, wherein one of the sbp members islabeled with a label that provides a detectable signal. The immunoassaymethodology results in a distribution of the signal label between signallabel bound in a complex of the sbp members and unbound signal label.The differentiation between bound and unbound signal label can be aresult of physical separation of bound from unbound signal label ormodulation of the detectable signal between bound and unbound signallabel.

In developing an assay device, there are many considerations. Oneconsideration is to provide substantial differentiation between theobserved signal resulting from signal label when bound as compared tounbound. Another consideration is the ease with which the observedsignal can be detected and serve to differentiate between the presenceor absence of analyte of interest. Other factors include the precisionwith which the test devices must be manufactured. Therefore, indeveloping an assay that can be used by untrained personnel, such asassays to be performed in the home, medical offices and the like, thetechnique for performing the assay should be simple, and the method ofmanufacturing the assay should be straightforward.

Plus/Minus Assays

Of particular interest to the present invention are test devices of thetype described in U.S. Pat. No. 5,145,789 to Corti et al., thedisclosure of which is incorporated herein by reference. Corti et al.discuss a built-in positive control to indicate successful operation ofa pregnancy test device. The positive control is envisaged as ahorizontal tract that always stains, independent of the presence of hCGin the urine, and is described as an area on a membrane that containsimmobilized hCG. Regardless of whether hCG is present in the biologicalsample, it is intended that during operation, the upstream mobilelabeled hCG binding reagents will always bind to the immobilized hCG,thereby forming a horizontal line, or minus sign, in the reading area.

A similar approach for providing a minus sign in a test device isdescribed in U.S. Pat. Nos. 4,916,056 and 5,008,080 to Brown, III etal., the disclosures of which are incorporated herein by reference. Asillustrated, the positive control is formed by providing a bindingsubstance within the test strip matrix, and is formed in the shape of arectangular bar, or minus sign. The binding substance of the minus signis intended to bind the labeled material regardless of the presence orabsence of the analyte of interest in the test sample.

Another approach for providing a positive control in a test device isdescribed in EP Patent Publication No. 0 249 418 to Graham, Jr., thedisclosure of which is incorporated herein by reference. As described,the control zone has anti-human IgG or IgM immobilized thereon, fornonspecifically capturing human immunoglobulin ubiquitously present inall similar human aqueous samples. The immobilized antibody is intendedto provide a signal in a “minus” pattern, regardless of the presence orabsence of the analyte of interest in the test sample.

Osikowicz et al., in U.S. Pat. No. 5,075,078, describe yet anotherapproach for providing a positive control in a plus/minus test device.The positive control is disposed on a test strip in a rectangular barconfiguration. The control bar is oriented on the strip so that it liesneither perpendicular nor parallel to the direction of fluid flow, butrather lies at an intermediate orientation, i.e., at a 45 degree angle.

Still yet another approach for providing a positive control in a testdevice is provided in U.S. Pat. No. 5,401,667 to Koike. As described,the test device provides a plus/minus format, but considers alternativegeometric symbols as well. A portion of the chromatographic medium isremoved, or otherwise partially blocked, thereby affecting the flow pathof the liquid. It is suggested that this modification enhances thesignal of the device.

Wong et al., in EP Patent No. 0 260 965, describe another test devicethat utilizes the plus/minus format. Wong et al. discuss a multiple-stepdiagnostic assay with a horizontal positive control line sprayed onto atest membrane.

The previous methods discussed above accomplish the “appearance” of aminus sign (−) by placing an indicator (positive control) lineperpendicular to the test line, directly onto the test strip. Typically,the control line develops with any sample flow, while the test linedevelops only with a positive sample flow. Thus, the previous assaysinvolve a control mechanism inherent to the matrix membrane test strip,and require a specific manufacturing step to apply the control line tothe strip.

Other previous devices display a printed minus sign positioned on thematrix and across the test line. These devices typically incorporate apositive control line downstream from, and parallel to, the test line.Such devices are limited as the test strips may present a line that isvisible before the sample is added.

Previous methods are further disadvantaged as the additionalmanufacturing step involves a difficult placement procedure to orientthe perpendicular line directly in the center of the viewing window.Whether the perpendicular line is a printed minus sign, or areagent-based control line, this approach is particularly ill suited forcertain matrix construction procedures, including web processing methodsthat involve a continuous flow or continuous roll application approach.

Therefore, it would be desirable to provide a test device that does notrequire this extra processing step of depositing a perpendicular lineonto the test strip, or does not leave a line that is visible before thesample is added to the device. This invention fulfills these and otherneeds.

Transparent Membranes

The use of transparent test strips in diagnostic assays is known in theart. In U.S. Pat. No. 4,824,640, Hildebrand et al. discuss a transparentreagent carrier layer suitable for evaluation by transmissionphotometry. As described, the transparent nature of the film of plasticprovides a suitable carrier material as compared to opaque films.

The use of a transparent test strip is also discussed in U.S. Pat. No.5,110,550 to Schlipfenbacher et al. As described, this test deviceincludes a white non-transparent covering layer situated above acolor-forming layer. During operation of the test device, the coveringlayer becomes transparent in the moist state. Through the transparentcovering layer, the user is then able to observe any reaction occurringin the color-forming layer below.

The use of a clearing agent in an immunochromatographic assay isdiscussed in U.S. Pat. No. 6,165,798 to Brooks. As described, the teststrip membrane is rendered transparent by wetting the membrane with aclearing agent, thus reducing the amount of light scattered by themembrane fibers.

In U.S. Pat. No. 6,187,268, Albarella et al. describe a transparent flowthrough membrane for use in test devices, but do not suggest a controlfeature to indicate a positive or negative test result. The membranedescribed in Albarella et al. is not configured to become transparentonly when wet. The membrane is transparent whether wet or dry.

While conceivably workable in some circumstances, the previous detectionsystems that employ transparent membranes are of limited utility. Thereis no teaching or suggestion in current art for a test device with atransparent membrane that utilizes a control feature to indicate apositive or negative test result as provided by a mark on the underlyingsupport.

In view of the foregoing, there remains a need in the art for a simple,efficient method for adding a positive control to a solid phase assaythat does not require the manufacturing step of fixing a positivecontrol binding member to the assay test strip, and that does not leavea substantially visible before the sample is added to the device. Itwould further be desirable to achieve improved test device formats thatincorporate transparent membranes as part of a control or displayfeature.

Additionally, the assay of the present invention should overcome thedisadvantages described above in connection with the previous testdevice systems.

BRIEF SUMMARY OF THE INVENTION

The present invention includes devices, methods, and kits for visuallydetecting the presence or absence of an analyte in a sample. Forexample, the present invention provides a test device for determiningthe presence or absence of an analyte in a fluid sample. The test devicecomprises a flow matrix disposed above a support. The matrix defines anaxial flow path, and typically contains three zones.

First, a sample receiving zone is located at an upstream end of the flowpath. The test sample suspected of containing an analyte of interest isapplied to the sample receiving zone. Second, a label zone is located onthe flow path downstream from the sample receiving zone. As the testsample flows through the label zone, it contacts a mobile labeledreagent. This labeled reagent is specific for, and binds to, the analyteof interest. Third, an observation area is located at the downstream endof the flow path. A capture zone is situated in the area, and containsimmobilized capture reagents that are specific for, and bind with, theanalyte of interest. Generally, the binding is achieved by either adirect or an indirect binding approach. Optionally, an absorbent zone isdisposed downstream of the observation area.

The sample fluid travels from the sample receiving zone, through thelabel zone, and then across the observation area and capture zone. Theobservation area, now moistened from the sample fluid, becomestransparent. The user can then view a mark displayed on the underlyingsupport.

The present invention is simple, effective, and solves the problems ofprevious devices and methods, by providing a test device with atransparent membrane and an underlying support that bears a mark.

This invention utilizes a property of nitrocellulose, or a similarmembrane, in that it becomes transparent when it is wet. By placing adetectable line or symbol directly underneath the nitrocellulose capturezone, that marking will be mostly invisible until a liquid sample isadded. When the membrane becomes wet, the markings underneath it becomeinstantaneously visible and appear to be a part of the displayedresults.

The invention described herein allows the mark to be placed in aprecisely controlled location within the window and can be printeddirectly on the plastic housing or on an intermediate member disposedbetween the housing and the test strip. This allows for the actual teststrip to be processed continuously in that there is no need tomanufacture a control line or minus symbol that is perpendicular to theprocessing flow. This feature is particularly desirable in webprocessing procedures.

The invention does not require any chemical interaction between a labelcomponent and a capture component to produce the appearance of a line orsymbol in any shape or color. The invention imparts a very distinct lookand operability characteristic to a lateral flow device.

The present invention achieves the “appearance” of a minus sign (−) byplacing an indicator mark perpendicular to the test line, directly onthe underlying support. The mark typically manifests with any sampleflow, while the test line develops only with a positive sample flow. Afurther advantage of the present invention is the avoidance of a linethat is substantially visible before the sample is added to the device.

Other objects and advantages will become apparent from the followingdetailed description taken in conjunction with the drawings in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded perspective view of a test device of the presentinvention;

FIG. 2 is an exploded perspective view of a test device of the presentinvention showing a negative test result;

FIG. 3 is an exploded perspective view of a test device of the presentinvention showing a positive test result; and

FIG. 4 is an exploded perspective view of a test device of the presentinvention showing a positive test result, and a procedural control line.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

In describing the various aspects of the present invention, a number ofterms will be generally defined or abbreviated as follows.

Definitions

“Fluid sample” refers to a material suspected of containing the analyteof interest. The fluid sample can be used as obtained directly from thesource or following a pretreatment so as to modify its character. Suchsamples can include human, animal or man-made samples. The sample can beprepared in any convenient medium which does not interfere with theassay. Typically, the sample is an aqueous solution or biological fluidas described in more detail below.

The fluid sample can be derived from any source, such as a physiologicalfluid, including blood, serum, plasma, saliva, sputum, ocular lensfluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid,peritoneal fluid, transdermal exudates, pharyngeal exudates,bronchoalveolar lavage, tracheal aspirations, cerebrospinal fluid,semen, cervical mucus, vaginal or urethral secretions, amniotic fluid,and the like. Herein, fluid homogenates of cellular tissues such as, forexample, hair, skin and nail scrapings, meat extracts and skins offruits and nuts are also considered biological fluids. Pretreatment mayinvolve preparing plasma from blood, diluting viscous fluids, and thelike. Methods of treatment can involve filtration, distillation,separation, concentration, inactivation of interfering components, andthe addition of reagents. Besides physiological fluids, other samplescan be used such as water, food products, soil extracts, and the likefor the performance of industrial, environmental, or food productionassays as well as diagnostic assays. In addition, a solid materialsuspected of containing the analyte can be used as the test sample onceit is modified to form a liquid medium or to release the analyte. Theselection and pretreatment of biological, industrial, and environmentalsamples prior to testing is well known in the art and need not bedescribed further.

“Specific pair binding member” refers to a member of a specific bindingpair, i.e., two different molecules wherein one of the moleculesspecifically binds with the second molecule through chemical or physicalmeans. The two molecules are related in the sense that their bindingwith each other is such that they are capable of distinguishing theirbinding partner from other assay constituents having similarcharacteristics. The members of the specific binding pair are referredto as ligand and receptor (antiligand), sbp member and sbp partner, andthe like. A molecule may also be a sbp member for an aggregation ofmolecules; for example an antibody raised against an immune complex of asecond antibody and its corresponding antigen may be considered to be ansbp member for the immune complex.

In addition to antigen and antibody specific binding pair members, otherspecific binding pairs include, as examples without limitation, biotinand avidin, carbohydrates and lectins, complementary nucleotidesequences, complementary peptide sequences, effector and receptormolecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes,a peptide sequence and an antibody specific for the sequence or theentire protein, polymeric acids and bases, dyes and protein binders,peptides and specific protein binders (e.g., ribonuclease, S-peptide andribonuclease S-protein), and the like. Furthermore, specific bindingpairs can include members that are analogs of the original specificbinding member, for example an analyte-analog or a specific bindingmember made by recombinant techniques or molecular engineering.

An sbp member is analogous to another sbp member if they are bothcapable of binding to another identical complementary sbp member. Suchan sbp member may, for example, be either a ligand or a receptor thathas been modified by the replacement of at least one hydrogen atom by agroup to provide, for example, a labeled ligand or labeled receptor. Thesbp members can be analogous to or complementary to the analyte or to ansbp member that is complementary to the analyte.

If the specific binding member is an immunoreactant it can be, forexample, an antibody, antigen, hapten, or complex thereof. If anantibody is used, it can be a monoclonal or polyclonal antibody, arecombinant protein or antibody, a chimeric antibody, a mixture(s) orfragment(s) thereof, as well as a mixture of an antibody and otherspecific binding members. The details of the preparation of suchantibodies and their suitability for use as specific binding members arewell-known to those skilled in the art.

When an immunoreactive specific binding member is attached to thechromatographic material of the present invention, the device isreferred to as an “immunochromatograph”, and the corresponding method ofanalysis is referred to as “immunochromatography”. Immunochromatography,as used herein, encompasses both sandwich and competitive immunoassaytechniques.

“Ligand” refers to any compound for which a receptor naturally exists orcan be prepared.

“Receptor” refers to any compound or composition capable of recognizinga particular spatial or polar orientation of a molecule, e.g., epitopicor determinant site. Illustrative receptors include: antibodies,enzymes, thyroxine binding globulin, intrinsic factor, lectins, nucleicacids, protein A, complement, complement C1q, and the like. Receptorsare also referred to as antiligands.

“Antigen” shall mean any compound capable of binding to an antibody, oragainst which antibodies can be raised.

“Antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof. Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon, and mu constant regions, as well as myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD, and IgE, respectively. Typically, an antibody is animmunoglobulin having an area on its surface or in a cavity thatspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of another molecule. Theantibody can be polyclonal or monoclonal. Antibodies may include acomplete immunoglobulin or fragments thereof. Fragments thereof mayinclude Fab, Fv and F(ab′)2, Fab′, and the like. Antibodies may alsoinclude chimeric antibodies made by recombinant methods.

“Analyte” or “analyte of interest” refers to the compound or compositionto be detected or measured and which has at least one epitope or bindingsite. The analyte can be any substance for which there exists anaturally occurring analyte specific binding member or for which ananalyte-specific binding member can be prepared. e.g., carbohydrate andlectin, hormone and receptor, complementary nucleic acids, and the like.Further, possible analytes include virtually any compound, composition,aggregation, or other substance which may be immunologically detected.That is, the analyte, or portion thereof, will be antigenic or haptenichaving at least one determinant site, or will be a member of anaturally-occurring binding pair.

Analytes include, but are not limited to, toxins, organic compounds,proteins, peptides, microorganisms, bacteria, viruses, amino acids,nucleic acids, carbohydrates, hormones, steroids, vitamins, drugs(including those administered for therapeutic purposes as well as thoseadministered for illicit purposes), pollutants, pesticides, andmetabolites of or antibodies to any of the above substances. The termanalyte also includes any antigenic substances, haptens, antibodies,macromolecules, and combinations thereof. A non-exhaustive list ofexemplary analytes is set forth in U.S. Pat. No. 4,366,241, at column19, line 7 through column 26, line 42, the disclosure of which isincorporated herein by reference. Further descriptions and listings ofrepresentative analytes are found in U.S. Pat. Nos. 4,299,916;4,275,149; and 4,806,311, all incorporated herein by reference.

“Label reagent” refers to a substance comprising a detectable labelattached with a specific binding member. The attachment may be covalentor non-covalent binding, but the method of attachment is not critical tothe present invention. The label allows the label reagent to produce adetectable signal that is related to the presence of analyte in thefluid sample. The specific binding member component of the label reagentis selected to directly bind to the analyte or to indirectly bind theanalyte by means of an ancillary specific binding member, which isdescribed in greater detail hereinafter. The label reagent can beincorporated into the test device at a site upstream from the capturezone, it can be combined with the fluid sample to form a fluid solution,it can be added to the test strip or device separately from the testsample, or it can be predeposited or reversibly immobilized at thecapture zone. In addition, the specific binding member may be labeledbefore or during the performance of the assay by means of a suitableattachment method.

“Label” refers to any substance which is capable of producing a signalthat is detectable by visual or instrumental means. Various labelssuitable for use in the present invention include labels which producesignals through either chemical or physical means. Such labels caninclude enzymes and substrates, chromogens, catalysts, fluorescentcompounds, chemiluminescent compounds, and radioactive labels. Othersuitable labels include colloidal metallic particles such as gold,colloidal non-metallic particles such as selenium or tellurium, dyed orcolored particles such as a dyed plastic or a stained microorganism,organic polymer latex particles and liposomes, colored beads, polymermicrocapsules, sacs, erythrocytes, erythrocyte ghosts, or other vesiclescontaining directly visible substances, and the like. Typically, avisually detectable label is used as the label component of the labelreagent, thereby providing for the direct visual or instrumental readoutof the presence or amount of the analyte in the test sample without theneed for additional signal producing components at the detection sites.

The selection of a particular label is not critical to the presentinvention, but the label will be capable of generating a detectablesignal either by itself, or be instrumentally detectable, or bedetectable in conjunction with one or more additional signal producingcomponents, such as an enzyme/substrate signal producing system. Avariety of different label reagents can be formed by varying either thelabel or the specific binding member component of the label reagent; itwill be appreciated by one skilled in the art that the choice involvesconsideration of the analyte to be detected and the desired means ofdetection. As discussed below, a label may also be incorporated used ina control system for the assay.

For example, one or more signal producing components can be reacted withthe label to generate a detectable signal. If the label is an enzyme,then amplification of the detectable signal is obtained by reacting theenzyme with one or more substrates or additional enzymes and substratesto produce a detectable reaction product.

In an alternative signal producing system, the label can be afluorescent compound where no enzymatic manipulation of the label isrequired to produce the detectable signal. Fluorescent molecules such asfluorescein, phycobiliprotein, rhodamine and their derivatives andanalogs are suitable for use as labels in such a system.

The use of dyes for staining biological materials, such as proteins,carbohydrates, nucleic acids, and whole organisms is documented in theliterature. It is known that certain dyes stain particular materialspreferentially based on compatible chemistries of dye and ligand. Forexample, Coomassie Blue and Methylene Blue for proteins, periodicacid-Schiff's reagent for carbohydrates, Crystal Violet, Safranin O, andTrypan Blue for whole cell stains, ethidium bromide and Acridine Orangefor nucleic acid staining, and fluorescent stains such as rhodamine andCalcofluor White for detection by fluorescent microscopy. Furtherexamples of labels can be found in, at least, U.S. Pat. Nos. 4,695,554;4,863,875; 4,373,932; and 4,366,241, all incorporated herein byreference.

“Signal producing component” refers to any substance capable of reactingwith another assay reagent or with the analyte to produce a reactionproduct or signal that indicates the presence of the analyte and that isdetectable by visual or instrumental means. “Signal production system”,as used herein, refers to the group of assay reagents that are needed toproduce the desired reaction product or signal.

“Ancillary specific binding member” refers to any member of a specificbinding pair which is used in the assay in addition to the specificbinding members of the conjugate or capture reagent. One or moreancillary specific binding members can be used in an assay. For example,an ancillary specific binding member can be capable of binding theconjugate with the analyte of interest, in instances where the analyteitself could not directly attach to the conjugate. Optionally, theancillary binding member may be capable of binding the analyte with thecapture reagent, in instances where the analyte itself could notdirectly attach to the capture reagent. The ancillary specific bindingmember can be incorporated into the assay device or it can be added tothe device as a separate reagent solution.

“Solid phase” refers to any solid material to which analyte, analytecomplexes, or assay reagents become bound and which can be separatedfrom unreacted assay reagents, test sample, or test solutions. Forexample, the solid phase may involve beads, magnetic particles, latexparticles, test tubes, microtiter plates, or any other solid material.Generally, the solid phase is any suitable chromatographic, bibulous,porous, isotropic, or capillary material, hereinafter referred to asporous material, which forms the basis of the test device.

“Capture reagent” refers to a specific binding member that is attachedwithin or upon a portion of the solid phase to form a “capture zone”.The method of attachment is not critical to the present invention. Thecapture reagent is selected to bind the analyte, the conjugate, or acomplex thereof.

“Ancillary material” shall mean any material that may be employed in anassay in accordance with the present invention. For example, buffer willnormally be present in the labeling means, the neutralization means, aswell as stabilizers for the assay medium and assay components.Frequently, in addition to these additives, additional protein, such asalbumin, or surfactant, non-ionic or ionic, binding enhancer, forexample, polyalkylene glycol, or the like, may be present, includingfree antibody, analyte analog, or other unrelated ligand, for thepurpose of removing or adding material or to modify the amount,position, partitioning, or appearance of the analyte or other compoundused in the invention.

Introduction

The present invention relates generally to devices and methods fordetection of analytes in test samples. More specifically, the presentinvention provides solid phase test strip devices and methods thatcombine an internal indicator on the test strip with an external marklocated on a support.

Test Device

In a first aspect, the present invention provides a test device fordetermining the presence or absence of an analyte in a fluid sample. Thetest device includes a support bearing a mark thereon, and a matrixdefining an axial flow path. Typically, the matrix further includes asample receiving zone, a label zone, and an observation area thatcontains a capture zone. In a related embodiment, the matrix furtherincludes an absorbent zone disposed downstream of the observation area.

In a preferred embodiment, the sample receiving zone accepts a fluidsample that may contain an analyte of interest. A label zone is locateddownstream of the sample receiving zone, and contains a mobile labelreagent that recognizes the analyte of interest. Further, an observationarea is disposed downstream of from the label zone, and contains animmobilized capture reagent that binds to the analyte of interest. Thus,as the fluid sample flows along the matrix, the analyte of interest willfirst bind with the mobile label reagent in the label zone, and thenbind to the immobile capture reagent in the capture zone of theobservation area. The observation area is made of a material that isopaque in a dry state and transparent in a moist state. Thus, the markon the support is visible through the observation area when theobservation area is in the moist state.

In another preferred embodiment, the fluid sample flows along a flowpath running from the sample receiving zone (upstream), through thelabel zone (midstream), and then to the observation area (downstream).Optionally, the fluid may thereafter flow to the absorbent zone.

In a preferred embodiment, the sample receiving zone is made of anabsorbent application pad. Suitable materials for manufacturingabsorbent application pads include, but are not limited to, hydrophilicpolyethylene materials or pads, glass fiber filter paper or pads,desiccated paper, paper pulp, fabric, and the like. In a relatedembodiment, the sample receiving zone is constructed from any materialthat absorbs water.

In a preferred embodiment, the absorbent application pad is made of anymaterial from which the fluid sample can pass to the label zone.Further, the absorbent application pad may be constructed to act as afilter for cellular components, hormones, particulate, and other certainsubstances that may occur in the fluid sample. Application pad materialssuitable for use by the present invention also include those applicationpad materials disclosed in U.S. Pat. No. 5,075,078, incorporated hereinby reference.

In yet another preferred embodiment, the absorbent application pad mayincorporate other reagents such as ancillary specific binding members,fluid sample pretreatment reagents, and signal producing reagents.

In a particularly preferred embodiment, the analyte of interest is fromthe group including heartworm antigens, hCG (human chorionicgonadotropin), streptococcus A, and other bacterial antigens.

In another preferred embodiment, the test device is configured toperform an immunological analysis process. In yet another embodiment,the liquid transport along the matrix is based upon capillary action,whereby the liquid transport path can be formed not only by one or morelayers of absorbent material, for example paper or fleece, but also by agap which is sucked full by capillary action.

In a preferred embodiment, the label zone is capable of non-bibulouslateral flow. By “non-bibulous lateral flow” is meant liquid flow inwhich all of the dissolved or dispersed components of the liquid arecarried at substantially equal rates and with relatively unimpaired flowlaterally through the membrane, as opposed to preferential retention ofone or more components as would occur, e.g., in materials capable ofadsorbing or imbibing one or more components.

In a further preferred embodiment, the label zone is made of a typicalnon-bibulous material such as high density polyethylene sheet materialmanufactured by Porex Technologies Corp. of Fairburn, Ga., USA. Thesheet material has an open pore structure with a typical density, at 40%void volume, of 0.57 gm/cc and an average pore diameter of 1 to 250micrometers, the average generally being from 3 to 100 micrometers. Theoptimum pore diameter for the membrane for use in the invention is about10 to about 50 μm. The membranes typically are from about 1 mil to about15 mils in thickness, typically in the range of from 5 or 10 mils, butmay be up to 200 mils and thicker. The membrane may be backed by agenerally water impervious layer, such as mylar. When employed, thebacking is generally fastened to the membrane by an adhesive, such as 3M444 double-sided adhesive tape. Typically, a water impervious backing isused for membranes of low thickness. A wide variety of polymers may beused provided that they do not bind nonspecifically to the assaycomponents and do not interfere with flow of the sample. Illustrativepolymers include polyethylene, polypropylene, polystyrene and the like.Alternatively, the membrane may be self supporting. Other non-bibulousmembranes, such as polyvinyl chloride, polyvinyl acetate, copolymers ofvinyl acetate and vinyl chloride, polyamide, polycarbonate, polystyrene,and the like, can also be used.

In yet another preferred embodiment, the label zone is made of amaterial such as untreated paper, cellulose blends, nitrocellulose,polyester, an acrylonitrile copolymer, and the like. The label zone maybe constructed to provide either bibulous or non-bibulous flow. In anespecially preferred embodiment, the label zone is made of a nonwovenfabric such as Rayon or glass fiber. Other label zone materials suitablefor use by the present invention include those chromatographic materialsdisclosed in U.S. Pat. No. 5,075,078, which is herein incorporated byreference. In a preferred embodiment, the label zone material may betreated with labeled solution that includes label reagents, blocking andstabilizing agents. Blocking agents include bovine serum albumin (BSA),methylated BSA, casein, nonfat dry milk. Stabilizing agents are readilyavailable and well known in the art, and may be used, for example, tostabilize colored labels.

In all of the above, employment of the selected blocking and stabilizingagents together with colored moieties in the labeling zone followed bythe immobilization of the blocking and stabilizing agents on the support(by, e.g., a freeze-drying process, or a forced air heat drying process)is of utmost importance for improved performance of the device. It iswell known that visible moieties, especially particles, aggregate uponair-drying and do not readily rehydrate in contact with a liquid sample.Therefore, absent conversion to the nonbibulous surface, instead ofbeing transported to the capture zone with the sample, the visiblemoieties will remain trapped in the labeling zone.

In a particularly preferred embodiment, the observation area will bemade of a material that is opaque when in a dry state, and transparentwhen in a moistened state, examples of which include nitrocellulose,nylon, and hydrophilic polyvinylidene difluoride (PVDF). Hydrophilicpolyvinylidene difluoride (PVDF) is commercially available form the firmMillipore, Bedford, U.S.A. under trademark Immobilon AV. However, on thebasis of the present description, the expert can also select othermaterials and especially synthetic material membranes which fulfill theabove-mentioned conditions. It is believed that the refractive index ofthe synthetic material is of major influence to this characteristic. Itis to be assumed that porous materials, the refractive index of which isclose to that of the sample liquid, have the property of becomingtransparent in a moist state.

In a particularly preferred embodiment, the observation area is anitrocellulose web assembly made of Millipore nitrocellulose rolllaminated to a clear Mylar backing. In another embodiment, theobservation area is made of nylon.

Those skilled in the art will appreciate that various methods could beused to test the desired property of the observation area, and whetherit is sufficiently opaque in the dry state, and sufficiently transparentin the moist state. For example, one could put a mark on a piece ofplastic, and then place the test strip on top of the plastic andvisually determine if it is opaque or transparent. Other methods includethe use of scanner devices, or other means for determining opticaldensity of the apparent line.

The artisan will appreciate the whole of the observation area, or only aportion thereof, may become transparent when moistened. In eitherembodiment, the portion of the observation area that is disposed abovethe mark should become transparent when moistened. In a relatedembodiment, the observation area will contain one or more distinct areasthat are opaque in the dry state, and transparent in the moist state.

In a preferred embodiment, the capture zone may be constructed from anyof the materials as listed above for the observation zone. In aparticularly preferred embodiment, the capture zone is made of the samematerial as the observation zone. The artisan will recognize that thepresent invention envisages a test device with one or more capturezones.

Further embodiments include capture zones that include microporousmaterials made from nitrocellulose, by which term is meant any nitricacid ester of cellulose. Thus suitable materials may includenitrocellulose in combination with carboxylic acid esters of cellulose.The pore size of nitrocellulose membranes may vary widely, but ispreferably within 5 to 20 microns, preferably 8 to 15 microns. Toprovide non-bibulous flow, these materials may be treated with blockingagents that can block the forces which account for the bibulous natureof bibulous membranes. Suitable blocking agents include bovine serumalbumin, methylated bovine serum albumin, whole animal serum, casein,and non-fat dry milk.

In a preferred embodiment, the observation area further includes aprocedural control line, to verify that the sample flow is as expected.The control line is a spatially distinct region that includes animmobilized binding member which reacts with a labeled reagent. In apreferred embodiment, the procedural control line contains an authenticsample of the analyte of interest, or a fragment thereof. In anotherpreferred embodiment, the control line contains antibody that isspecific for, or otherwise provides for the immobilization of, thelabeled reagent. In operation, a labeled reagent binds to the controlline, even when the analyte of interest is absent from the test sample.

In a related embodiment, a control conjugate is introduced into the flowsample upstream from the control line. For example, the controlconjugate may be added to the fluid sample before the sample is appliedto the assay device. Alternatively, the control conjugate may bediffusively bound in the sample receiving zone, or in the label zone.

In a preferred embodiment, the control conjugate includes a controllabel and a control reagent. Typically, a control reagent is chosen tobe different from the reagent that is recognized by the capture reagent.Further, the control agent is generally not specific for the analyte. Ina preferred embodiment, the control reagent binds to a control capturepartner that is immobilized on the procedural control line. Thus thecontrol conjugate is directly detected in the control line.

In yet another embodiment, the detectable control label is the samelabel used for the label reagent. In a preferred embodiment, the controllabel is different from the test label, so that the user can more easilydetermine the results of the assay. In a particularly preferredembodiment, the control label and the test label include colored beads,where the control and test beads are of different colors.

In a preferred embodiment, the control label includes streptavidin, andthe control capture partner includes biotin, which couples to the avidinspecifically. In a particularly preferred embodiment, the control labeincludes biotin, and the control capture partner includes streptavidin.The artisan will appreciate that other “irrelevant” binding pairs canalso be used-such as antigen/antibody reactions unrelated to analyte.

The use of a control line is helpful in that appearance of a signal inthe control line indicates the time at which the test result can beread, even for a negative result. Thus, when the expected signal appearsin the control line, the presence or absence of a signal in the capturezone can be noted.

In another preferred embodiment, the matrix may further incorporate anabsorbent zone. The absorbent zone can act to increase the amount offluid sample that travels through the capture zone.

In this embodiment, the absorbent zone is located downstream from thecapture zone and can be a means for removing excess sample and freelabel other than the analyte of interest from the matrix of the device.Generally, the absorbent zone will consist of an absorbent material suchas filter paper, a glass fiber filter, or the like.

In a preferred embodiment, the device may also contain an end of assaycontrol zone indicator. The control zone indicator may consist of a pHindicating reagent (such as bromocresol green) impregnated in theabsorbent zone or at a location downstream of the capture zone. Uponcontact with the sample, a pH change occurs in the processed matrix.This pH shift converts the pH indicator to a different color (forinstance, bromocresol green may be converted from yellow to blue) whichis seen in an observation window over the control zone. This technologymay also serve as an internal assay control.

In a related embodiment, the end of assay control zone may beconstructed by applying a line of soluble ink on the capture zone (atthe interface with the absorbent zone). The liquid front moving throughthe capture zone will solubilize the ink and transfer it into theabsorbent. The resulting color change will be seen in an observationwindow above the absorbent zone, signifying end of assay.

In a preferred embodiment, the capture reagent binds with the analytefor the completion of a sandwich complex. The capture reagent can bechosen to directly bind the analyte or indirectly bind the analyte bybinding with an ancillary specific binding member which is bound to theanalyte. In addition, the capture reagent may be immobilized on thesolid phase before or during the performance of the assay by means ofany suitable attachment method. Typically, the capture site of thepresent invention is a delimited or defined portion of the solid phasesuch that the specific binding reaction of the capture reagent andanalyte is localized or concentrated in a limited site, therebyfacilitating the detection of label that is immobilized at the capturesite in contrast to other portions of the solid phase. In a relatedembodiment, the capture reagent can be applied to the solid phase bydipping, inscribing with a pen, dispensing through a capillary tube, orthrough the use of reagent jet-printing or other techniques. Inaddition, the capture zone can be marked, for example with a dye, suchthat the position of the capture zone upon the solid phase can bevisually or instrumentally determined even when there is no labelimmobilized at the site.

Those of skill in the art will recognize that a variety of direct andindirect assay formats may be employed in the present invention. In apreferred embodiment, a direct assay format is used. Direct assays areexemplified by those that detect the presence of an antigen in a sample,as well as those that detect the presence of an antibody in a sample. Adirect assay that detects the presence or absence of an antigen in asample generally includes a label zone that contains at least oneantibody label reagent that is specific for the antigen. In a preferredembodiment, the antibody is coated on colored beads. Likewise, a directassay that detects the presence or absence of an antigen in a samplegenerally includes a label zone that contains at least one antigen thatis reactive with an antibody-analyte of interest. In this embodiment,the antigen is coated on colored beads.

In a preferred embodiment, the result displayed by the test deviceincorporates a symbol provided by the mark on the bottom support. Due tothe transparent nature of the observation area, the mark on the supportis detectable through the moistened observation area. Thus, the shape ofthe mark will be displayed regardless of whether the analyte of interestis or is not present in the fluid sample. Typically, the shape of themark provides a first symbol to represent a negative result. In arelated embodiment, the symbol provided by the mark on the support is aminus (−) symbol. In a particularly preferred embodiment, the mark willbe visible to the unaided eye, although those of skill in the art willappreciate that a variety of detection approaches may be employed. Inanother preferred embodiment, the mark will be blue or red, or any colorwhich provides contrast with the surrounding area, which is typically alighter color, such as white.

In a preferred embodiment, the mark on the support will operate inconcert with a symbol provided by the capture zone. When the desiredanalyte is present in the fluid sample, label is collected at thecapture zone. The shape of the labeled capture zone is such that, incombination with the mark, a second symbol is provided representing apositive result. In a related embodiment, the symbol presented by thiscombination is a plus (+) symbol.

In yet another preferred embodiment, the capture zone has a shape suchthat, independent of any symbol provided by that mark on the substrate,labeled reagent at the capture zone will form a second symbol torepresent a positive test result.

In a preferred embodiment, the mark on the support is oriented so thatthe longitudinal axis of the mark is aligned with the direction of thesample flow, while the longitudinal axis of the capture zone isperpendicular to the direction of the sample flow. In a relatedembodiment, these axes may be at oblique angles to the sample flow. Inyet another related embodiment, these axes may be oriented at any angleto the sample flow, so long as the axis of the mark and the axis of thecapture zone are substantially perpendicular to each other.

In another preferred embodiment, the mark on the support includes ashape such that, in the presence or absence of the analyte of interestin the fluid sample, a logo is displayed in the observation area. In arelated embodiment, the marked support provides one or more predefinedsymbols to be displayed when the observation area is moistened. Thesesymbols may be read in connection with, or independent from, any signalthat may be provided by the capture zone.

In yet another embodiment, the mark may be displayed below the capturezone, but not necessarily on the bottom support. For example, the markmay be contained on the Mylar strip underlying the nitrocellulose matrixmembrane. Alternatively, the mark may be contained on an intermediatemember disposed between the test strip and the bottom support. In apreferred embodiment, the intermediate member is a plastic stripdisplaying the mark. The member may or may not be affixed to either thetest strip or the bottom support. In a preferred embodiment, theintermediate member is configured to remain in place once the testapparatus is constructed. In a related embodiment, the mark is applied,for example, by an embossing, printing, or stamping technique.

In still another preferred embodiment, the observation area includes anegative control area. The purpose of this control area is to alert theuser that the test device is not working properly. In a preferredembodiment, the negative control is that part of the observation areaoutside of the capture zone, and does not include any part of theobservation area located directly above the mark on the support. Whenworking properly, no signal or mark should be visible in the negativecontrol area.

In a preferred embodiment, the test device includes a hollow casing orhousing having an application aperture and an observation port. In thisembodiment, the flow matrix is contained within the hollow casing, andthe fluid sample is added to the matrix through the aperture, which isan opening located in an upstream location on the housing.

Typically, the aperture is located above the sample application pad. Ina related embodiment, an aperture may be disposed in any location abovethe matrix that would provide for facile addition of fluid sample orreagent to the matrix.

To detect the result, the observation area may be examined through theport. The artisan will appreciate that an observation port may bedisposed in any location above the matrix that would provide for thedetection of a signal event, whether the signal comes from theobservation area, the absorbent zone, or elsewhere. In a preferredembodiment, the port is located above the observation area. Generally,the observation port is open, or the port may be covered by atransparent covering such as glass or plastic.

From the foregoing, it is appreciated that the outer casing or housingof the device may take various forms. Typically, it will include anelongate casing and may have a plurality of interfitting parts. In aparticularly preferred embodiment, the housing includes a top cover anda bottom support. In a preferred embodiment, the bottom support displaysa mark thereon, and in a related embodiment, the top cover contains anapplication aperture and an observation port. In another embodiment, thehousing may also contain dividers between the matrix strips to inhibitflow of fluid sample between strips.

In a preferred embodiment, the housing is made of moisture impervioussolid material, for example, a plastic material. It is contemplated thata variety of commercially available plastics, including, but not limitedto, vinyl, nylon, polyvinyl chloride, polypropylene, polystyrene,polyethylene, polycarbonates, polysulfanes, polyesters, urethanes, andepoxies maybe used to construct a housing. The housing may be preparedby conventional methodologies, such as standard molding technologiesthat are well known and used in the art. The housing may be produced bymolding technologies which include, but are not limited to, injectionmolding, compression molding, transfer molding, blow molding, extrusionmolding, foam molding, and thermoform molding. The aforementionedmolding technologies are well known in the art and so are not discussedin detail herein. See for example, Processes And Materials OfManufacture, Third Edition, R. A. Lindsberg (1983) Allyn and Baron pp.393-431.

It will be appreciated by one skilled in the art that a test stripdevice can be made of more than one material (e.g., different zones orsites can be made of different materials) and a flow-through device canhave more than one layer, wherein different layers can be made ofdifferent materials, so long as the multiple materials or layers are influid-flow contact with one another thereby enabling the passage of testsample between the materials or layers. Fluid-flow contact permits thepassage of at least some components of the test sample between the zonesor layers of the device. Fluid-flow is preferably uniform along thecontact interface between the different zones or layers. The presentinvention further provides for different reagents disposed on differentmaterials, and different reagents disposed on different zones. Forexample, the specific binding member contained on a certain material inthe label zone may or may not be the same binding member that iscontained on a different material in the capture zone.

In further preferred embodiments, the test device of the presentinvention can have many configurations, several of which are dependentupon the material chosen for the solid phase. For example, the testdevice can include a solid phase material configured for use in alayered flow-through assay device, a chromatographic column, a dipstickor a test strip.

In a second aspect, the present invention provides a method fordetermining the presence or absence of an analyte in a fluid sample. Theassay method includes providing a test device as described above, andadministering a fluid sample to the test device. By inspecting theobservation area of the test device, the user is able to determinewhether the fluid sample contains the analyte of interest. Typically,this is accomplished by observing either a plus (+) or minus (−) signalin the observation area.

In a third aspect, the present invention provides a kit for determiningthe presence or absence of an analyte in a fluid sample. The assay kitof the present invention will include containers for holding anynecessary reagent, test membrane, sample applicator, filter, and thelike, as well as instructions which set forth a protocol for practicingthe method of use as described above. Typically, the components of thekit will be present in a suitable package containing sufficientcomponents for performing one or more assays.

The present invention is particularly suitable for a test device asshown in the accompanying drawings, and described in detail as follows.It is understood that the drawings are provided for purposes ofillustration and not meant limit the scope of the present invention.

FIG. 1 shows a first embodiment of a test device 10 constructed inaccordance with the teachings of the present invention.

Test device 10 has a bottom support 14, a flow matrix 18, a top cover22, and a desiccant 26. In its longitudinal direction, matrix 18 can besubdivided into a sample application zone 30, a label zone 34, anobservation area 38, and an absorbent zone 42.

The bottom support further includes a mark 46 thereon. The mark isdisposed beneath observation area 38. The sample application zone islocated at an upstream location on matrix 18, and is configured toreceive the fluid sample. Label zone 34 is located downstream ofapplication zone 30, and contains label reagent. The observation area islocated downstream of the label zone, and includes a capture zone 40that contains capture reagent. Absorbent pad 42 is located downstream ofobservation area 38.

Top cover 22 has an application aperture 48 disposed above the sampleapplication pad, and an observation port 52 disposed above theobservation area. In cooperation, the top cover and the bottom supportare configured to provide a housing for matrix 18 and desiccant 26. Asshown, the desiccant is typically positioned separately from the matrix.

Prior to use, when in the dry state, the observation area is opaque, andthus no signal is manifested through the observation port.

FIG. 2 illustrates the test device of the present invention showing anegative test result, in the instance where the fluid sample does notcontain the analyte of interest.

In operation, the sample fluid is added through aperture 48, and on toapplication pad 30. The fluid sample is transported from application pad30 to label zone 34, where the fluid is allowed to react with labeledreagent. Labeled reagent then binds with any analyte of interestcontained in the fluid sample.

Next, the fluid sample is advanced to observation area 38, and then onto the absorbent zone. Observation area 38, now moistened by the samplefluid, becomes transparent. Consequently, mark 46 becomes visiblethrough observation port 52. Through the observation port, the user canthen view the mark displayed on the bottom support.

As shown here, there is no analyte present, and consequently no label isbound at capture zone 40. The resulting signal as manifested throughobservation port 52 is therefore in the shape of a minus (−) sign.

FIG. 3 illustrates the test device of the present invention showing apositive test result, in the instance where the fluid sample containsthe analyte of interest.

In operation, the sample fluid is added through aperture 48, and on toapplication pad 30. The fluid sample is transported from application pad30 to label zone 34, where the fluid is allowed to react with labeledreagent. Labeled reagent then binds with the analyte of interestcontained in the fluid sample. Next, the fluid sample is advanced toobservation area 38, and labeled analyte is restrained by capturereagent immobilized in capture zone 40. Unbound material in the fluidsample continues to flow into absorbent zone 42.

Observation area 38, now moistened by the sample fluid, becomestransparent. Consequently, mark 46 becomes visible through observationport 52. Through the observation port, the user can then view the markdisplayed on the bottom support, in concert with the signal provided bythe label contained in the capture zone. The resulting signal asmanifested through observation port 52 is in the shape of a plus (+)sign.

FIG. 4 shows the test device of the present invention, where observationarea 38 further includes a procedural control line 56 that develops withany fluid sample flow, whether or not analyte is present in the sample.

The invention can be better understood by way of the following examplewhich is representative of the preferred embodiment, but is not to beconstrued as limiting the scope of the invention.

EXAMPLES Example 1

A lateral flow test device was constructed according to principles ofthe present invention. The test device included a G-III plastic bottom(#Z0846500), a G-III plastic scanner top (#Z0440900), a sample padcontaining stock assay reagent, a label pad containing stock assayreagent, an observation zone as described below, and an absorbent pad(#0841000).

A nitrocellulose web assembly membrane from Millipore, trade nameHighflow Plus Membrane, was combined with a clear Mylar backing.

A dark line was made on the inside of the plastic bottom, positionedparallel to sample flow in approximately the same area as would beunderneath the fixed minus line from a current assay strip.

This bottom was then assembled with an experimental G-IV assay strip asdescribed above, and a plastic scanner top. When a liquid sample wasadded to the test unit, the dark line, which was barely visible throughthe dry strip, became clearly visible. The results are shown generallyin FIG. 2.

Example 2

Goat anti-alpha hCG antibodies were immobilized on Hi-flow plusnitrocellulose membrane (nitrocellulose membrane cast on a transparentnylon sheet), manufactured by Millipore Inc. A second, unrelated proteinwas immobilized on the membrane for the procedural control line. Themembrane was then blocked with a protein solution and dried prior toassembly.

A sample pad (non-woven rayon fiber backed with mylar) was impregnatedwith a buffered protein solutions and dried.

The label pad (non-woven rayon fiber backed with mylar) was impregnatedwith a solution containing red colored polystyrene microspheres coatedwith anti-beta hCG monoclonal antibodies, blue colored polystyrenemicrospheres coated with a binding pair member to the control lineprotein, and stabilizing agents followed by a drying process.

The sample and label pad, the capture membrane and an absorbent pad werethen assembled into a test strip similar to FIG. 4.

A visible line was printed onto the bottom half of the plastic housingof a test cassette. The line was placed directly beneath andperpendicular to the immobilized goat anti-alpha hCG test line on thecapture membrane. The preprinted line was approximately 1 mm thick and5-9 mm long. The assembled test strip was then placed on top of thepreprinted line into the bottom of the plastic housing as indicated inFIGS. 1-4, and the cassette was closed with the top portion of theplastic housing. Upon assembly, the preprinted line on the plastichousing was nearly invisible.

Upon addition of a liquid sample, the sample pad and label pad wererehydrated and the sample wet the nitrocellulose capture membrane thusmaking it transparent. The pre-printed line which was barely visible inthe dry state was now clearly visible. The results are shown generallyin FIG. 4.

1. A test device for determining the presence or absence of an analytein a fluid sample, the test device comprising: a) a solid support; b) anintermediate piece bearing a mark thereon; c) a matrix defining an axialflow path, the matrix comprising: i) a sample receiving zone, at anupstream end of the flow path, that receives the fluid sample, ii) alabel zone, on the flow path downstream from the sample receiving zone,comprising a label reagent that binds with the analyte in the fluidsample flowing through the label zone, and iii) an observation area, onthe flow path downstream from the label zone, the observation areacomprising a defined capture zone having an immobilized capture reagentthat restrains the analyte, wherein the observation area comprises amaterial that is opaque in a dry state and transparent in a moist stateand the mark on the intermediate piece is visible through theobservation area when the observation area is in the moist state;wherein the intermediate piece is disposed between the matrix and thesupport, iv) the mark has a shape such that, in the absence of theanalyte in the sample, a first symbol is displayed to represent anegative result, and v) the capture zone has a shape such that, in thepresence of analyte in the sample and taken together with the firstsymbol, a second symbol is formed to represent a positive result.
 2. Thetest device of claim 1, wherein the first symbol is in the shape of aminus symbol and the second symbol is in the shape of a plus symbol. 3.The test device of claim 2, wherein the longitudinal axis of the mark isin the direction of the sample flow, and the longitudinal axis of thecapture zone is perpendicular to the direction of the sample flow. 4.The device of claim 1, wherein the first symbol is a logo.
 5. The testdevice of claim 1, wherein the first symbol comprises one or morepredefined symbols.
 6. The test device of claim 1, wherein theobservation area further comprises a negative control area.
 7. The testdevice of claim 1, wherein the observation area further comprises apositive control area.
 8. The test device of claim 1, further comprisinga hollow casing having a sample application aperture and an observationport, wherein the matrix is contained within the hollow casing, thesample is applied to the matrix through the aperture, and theobservation area can be observed through the port.
 9. The test device ofclaim 8, wherein the observation port is covered by a transparentcovering.
 10. A method of manufacturing the test device of claim 1, themethod comprising: a) providing a solid support; b) providing anintermediate piece bearing a mark thereon; c) providing a matrixdefining an axial flow path, wherein the matrix comprises: i) a samplereceiving zone, at an upstream end of the flow path, that receives thefluid sample, ii) a label zone, on the flow path downstream from thesample receiving zone, comprising a label reagent that binds with theanalyte in the fluid sample flowing through the label zone, and iii) anobservation area, on the flow path downstream from the label zone, theobservation area comprising a capture zone having an immobilized capturereagent that restrains the analyte, and wherein the observation areacomprises a material that is opaque in a dry state and transparent in amoist state and the mark on the intermediate piece is visible throughthe observation area when the observation area is in the moist stateand; d) assembling the test device such that the intermediate piece isdisposed between the matrix and the solid support.
 11. A test device fordetermining the presence or absence of an analyte in a fluid sample, thetest device comprising: a) a solid support; b) an intermediate piecebearing a mark thereon; c) a matrix defining an axial flow path, thematrix comprising: i) a sample receiving zone, at an upstream end of theflow path, that receives the fluid sample, ii) a label zone, on the flowpath downstream from the sample receiving zone, comprising a labelreagent that binds with the analyte in the fluid sample flowing throughthe label zone, and iii) an observation area, on the flow pathdownstream from the label zone, the observation area comprising adefined capture zone having an immobilized capture reagent thatrestrains the analyte, wherein the observation area comprises a materialthat is opaque in a dry state and transparent in a moist state and themark on the intermediate piece is visible through the observation areawhen the observation area is in the moist state, wherein theintermediate piece is disposed between the matrix and the support, andiv) the mark has a shape such that, in the presence or absence of theanalyte in the sample, a first symbol is displayed, and v) the capturezone has a shape such that, independent of the first symbol, in thepresence of the analyte in the sample, a second symbol is formed torepresent a positive result.
 12. The test device of claim 11, whereinthe first symbol is a logo.
 13. The test device of claim 11, wherein thefirst symbol comprises one or more predefined symbols.
 14. The testdevice of claim 11, wherein the observation area further comprises anegative control area.
 15. The test device of claim 11, wherein theobservation area further comprises a positive control area.
 16. The testdevice of claim 11, further comprising a hollow casing having a sampleapplication aperture and an observation port, wherein the matrix iscontained within the hollow casing, the sample is applied to the matrixthrough the aperture, and the observation area can be observed throughthe port.
 17. The test device of claim 16, wherein the observation portis covered by a transparent covering.
 18. A method of manufacturing thetest device of claim 11, the method comprising: a) providing a solidsupport; b) providing an intermediate piece bearing a mark thereon; c)providing a matrix defining an axial flow path, wherein the matrixcomprises: i) a sample receiving zone, at an upstream end of the flowpath, that receives the fluid sample, ii) a label zone, on the flow pathdownstream from the sample receiving zone, comprising a label reagentthat binds with the analyte in the fluid sample flowing through thelabel zone, and iii) an observation area, on the flow path downstreamfrom the label zone, the observation area comprising a capture zonehaving an immobilized capture reagent that restrains the analyte, andwherein the observation area comprises a material that is opaque in adry state and transparent in a moist state and the mark on theintermediate piece is visible through the observation area when theobservation area is in the moist state; and d) assembling the testdevice such that the intermediate piece is disposed between the matrixand the solid support.